Tumor cells are often present in blood of carcinoma patients as rare cells at very low frequencies (<10 cells per ml). These levels may provide clinically useful information. However, the laborious procedures required to detect and quantify the presence of circulating tumor cells and the statistics of low numbers introduce a high level of variability in the results. The system of this invention improves upon imaging devices used in a clinical laboratories for rare cell detection in fluid samples such as blood samples (i.e. circulating rare tumor cells of epithelial origin).
Generally, these rare cells are targeted by labeling and separating from blood by magnetic means. The captured cells are fluorescently labeled to permit detection and differentiation from non-target cells and control cells. CellSpotter® is a semi-automated fluorescence microscope that enumerates and differentiates between immuno-magnetically selected cells based upon fluorescence signals from cells in the blood sample.
A blood sample, suspected of containing the targeted rare cells, is preprocessed by enriching the desired rare cells. Enrichment is obtained by linking antibodies, specific for the target cell, to magnetic particles. This immunomagnetic complex is combined with the blood sample in the presence of a magnetic field, providing for the immunomagnetic capture of the target cells. After obtaining an enriched blood fraction containing the target cell, fluorescent reagents are added for subsequent imaging. Enriched cells are placed into a viewing cartridge which then is placed into a magnetic device that directs magnetically labeled cells in the sample to an optically clear planar surface of the chamber for immuno-magnetical alignment and image analysis. The novel sample chamber (U.S. application Ser. Nos. 10/074,900 and 10,303,309) used in the present invention and the Cell Spotter® system are incorporated in the following patents and co-pending applications; U.S. Pat. No. 5,186,827, 5,698,271, 6,120,856, 6,551,843, U.S. application Ser. Nos. 09/702,188, 10/449,355. The magnetic device and chamber are then placed on a fluorescence microscope equipped with a computer controlled filter selector and digitally controlled X-Y-Z stage. Images are acquired by scanning the sample cartridge with 4 different fluorescent filter sets. The acquired images are processed by automated software analysis to compile data containing images of the target cells. Individual frames are viewed and manually selected for target cells. The analysis is complete with a presentation of a gallery of images containing all selected target cells.
When incorporated into a clinical laboratory, the system can be used to examine circulating rare cells associated with clinical disease. For example, multi-center prospective, longitudinal clinical trials can be redially established whereby the number of circulating tumor cells (CTC) are correlated with disease progression in patients having metastatic breast cancer (U.S. Pat. Nos. 6,365,362; 6,645,731; U.S. application Ser. No.10/079,939).
Thus, the principal concepts of the present invention stem from laboratory diagnostic equipment systems assessing the detection and enumeration of cells in biological specimens, together with microscope systems for observing the microstructure of a cell under a desired magnification.
Microscopy technology employing stepper motor driven stages for imaging areas larger than the microscope field of view has limited clinical utility. For example, the achievable speed of image acquisition in image cytometry analysis and the overall lab bench space required for the CellSpotter® System limits it practical clinical use. Currently, image acquisition is accomplished by moving a slide or sample cartridge through the field of view in a boustrophedonic step-by-step motion. A boustrophedonic motion is a motion whereby two consequtive lines along the fastest axis are scanned in opposite directions. One image is taken for each step. When multiple fluorescent images of the same sample are obtained, the filter cube is changed and the boustrophedonic motion is repeated after completion of each pass. This process is repeated until an image is acquired for all positions and all colors, completing a color-by-color scan. Both the movement of the filter cube and the sample is accomplished by stepper motor stages with servo feedback, severly limiting the acquisition speed. Moving a filter cube to the next takes approximately 2 seconds with an additional 0.5 seconds to step between two consecutive positions. Thus, the total acquisition time consists of the total motion time plus the total image acquisition time, making acquisition the predominant time spent on motion with imaging systems like CellSpotter®.
Because the prior art employs stepper motor technology for driving microscope stages which is limited to a maximum driving frequency on the stepper motors, there is a need to develop new devices to improve upon large area image acquisition time. Furthermore, moving the sample (or cartridge) can cause the cells to move with the inertia of the fluid. Any motion of a cell could cause the acquisition software to misinterpret the images and count one cell as two with unpredictable identification.
Another issue with the prior art is that during stage motion the sample is continually being illuminated. Consequently, fluorescent dyes used in cell labeling will undergo bleaching. Phycoeurythin (PE) is a dye comonly used in cell labeling, but very sensitve to bleaching. Reducing the time spent on motion will reduce the extent of sample bleaching.
The equipment encompassed in CellSpotter® technology includes a sample chamber (U.S. application Ser. Nos. 10/074,900, 10/303,309, and U.S. Pat. No. 5,993,665) is placed into a magnetic device (U.S. Pat. No. 5,985,153) that directs magnetically labeled cells in the sample to an optically clear planar surface of the chamber. The magnetic device and chamber are then placed on a fluorescence microscope equipped with a computer controlled filter selector and digitally controlled X-Y-Z stage.
The present invention is an improvement to the CellSpotter® automated diagnostic system to provide rapid sample analysis using multiple fluorescent indicators. The imaging device is further improved by condensing the components into a simple box shape for convienent placement on most clinical laboratory bench tops. This providing a practical configuration in automated laboratories that lack substantial amout of space.